人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏
Summary
本文介绍了一个协议的 large-scale 无隔离和收集自由毛蚴的人类血液侥幸血吸虫氏及其后续的处理, 介绍到体外文化。
Abstract
人的血液吸虫,血吸虫, 有一个复杂的生命周期, 涉及无性和性发育阶段内的蜗牛中间和哺乳动物的最终宿主, 分别。在体外培养条件下, 分离和维持不同生命周期阶段的能力大大促进了对寄生生物生长、发育和寄主的细胞、生物化学和分子机制的研究。相互.血吸虫病的传播需要在钉螺寄主内无性繁殖和发育多个幼体期;从感染毛蚴, 通过初级和次级 sporocysts, 到最后的尾蚴阶段, 是感染人类。本文提出了一个 step-by 的程序, 用于大规模孵化和隔离的血吸虫氏毛蚴从被感染小鼠的肝脏获得的卵子, 并随后介绍到体外培养。预计详细的协议将鼓励新的研究人员参与并拓宽这一重要的血吸虫研究领域。
Introduction
人的血液吸虫血吸虫, 是血吸虫病的致病因子, 一种被忽视的热带疾病, 在全世界估计有2.3亿人在1。最广泛的物种,日本血吸虫氏, 分布在南美洲, 加勒比群岛, 中东和撒哈拉以南非洲地区2。S. 氏和其他血吸虫的生命周期是复杂的, 涉及哺乳动物的确定性宿主和Biomphalaria的淡水蜗牛, 它们充当强制的中间宿主。
S. 氏雄性和雌性成年蠕虫对生活在哺乳动物宿主的后肠系膜静脉中, 导致胚卵 (卵) 在小肠黏膜的小静脉中释放。卵然后冲破血管壁, 通过粘膜组织迁移, 最终进入肠道, 在那里它们被粪便所破坏。当卵进入淡水和自由幼虫 (毛蚴) 孵化, 他们必须, 在短的顺序, 找到一个合适的Biomphalaria蜗牛感染, 以继续其生命周期。这一感染过程涉及 miracidial 附着在蜗牛的外体表, 其次是主动渗透和进入寄主的幼虫。进入后不久, 毛蚴开始脱落其外纤毛表皮板, 因为它形成了一个合, 将成为新的外表面 (皮) 的下一个幼虫阶段, 主要或母亲孢子囊。通过无性繁殖, 每个主要孢子囊生产和释放第二代 sporocysts, 称为次要或女儿 sporocysts, 反过来, 生成和释放大量的最终 intramolluscan 阶段, 尾蚴3.从蜗牛主机逃生后, 自由尾蚴能够附着并直接穿透人类或其他合适的哺乳动物宿主的皮肤。进入新寄主后, 尾蚴转化为寄生童阶段, 侵入血管系统。反过来, 他们迁移到肺, 然后到肝静脉, 他们成熟的成年蠕虫, 形成男性和女性对和旅行到肠系膜静脉, 以完成他们的生命周期。
成功的 intramolluscan 或 “蜗牛相” 发育的幼虫血吸虫是必不可少的继续循环的人类主机传播。最重要的是自由毛蚴进入蜗牛寄主的时期, 并将其早期转化为初级孢子囊阶段。根据寄主和寄生虫之间的生理和免疫相容性, 这是在幼体发育的这一阶段, 最初的成功或失败建立感染是确定的4,5,6.随后开发的初级 sporocysts 能够无性生产的次生 sporocysts, 然后产生人感染尾蚴, 进一步需要一个宽容的生理宿主环境, 提供所有的寄生虫的增长和生殖需求。
到目前为止, 关于控制血吸虫-蜗牛相互作用的基本生理、生物化学和分子过程, 特别是有关调节幼虫发育和繁殖的分子和途径, 或调节宿主免疫应答。在体外条件下, 可随时访问大量这些幼体阶段, 允许进行实验操作的培养基将极大地促进发现发育途径和细胞生长的基本机制, 分化和繁殖。关键的寄生虫通路或机制, 通过体外试验确定, 然后可以用来扰乱幼虫生长/繁殖在蜗牛寄主, 或识别蜗牛宿主免疫机制参与识别和消除miracidial 或孢子囊阶段。
在本文和视频演示中, 我们提供了一种方法的详细描述, 用于隔离大量的自由S. 氏毛蚴, 以便导入到体外区域性, 然后再进行后续实验操作 (有关协议工作流的示意图概述, 请参见图 1 )。虽然先前已描述过类似的过程7、8、9, 但我们认为, 详细的协议对于希望使用此模型来解决与intramolluscan 幼虫发育, 细胞增殖, 血吸虫-蜗牛免疫相互作用。此外, 这种方法可以很容易地适应分离的卵子从其他医学重要的吸, 如膀胱居住的S. 埃及, 肝吸虫或肺吸虫。
Protocol
Representative Results
Discussion
毛蚴的S. 氏的分离和操作, 如本文所述, 只能感染蜗牛中间宿主, 因此在幼体发育的这一阶段不代表人类的生物危害。然而, 为了避免意外暴露/感染蜗牛, 应注意执行 miracidial 隔离在不同的地方, 在敏感的Biomphalaria蜗牛物种可能存在或维持。一个独立的房间, 注册为 BSL2 空间, 是强烈建议。此外, 在处理肝脏和幼体隔离过程中使用的所有洗涤液都应使用消毒剂 (如, 1% 漂白剂) 处理。…
Divulgations
The authors have nothing to disclose.
Acknowledgements
部分由 NIH 资助 RO1AI015503。血吸虫感染小鼠由 NIAID 血吸虫病资源中心在生物医学研究所 (马里兰州罗克韦尔, MD) 通过 NIH-NIAID 合同 HHSN272201000005I 提供通过北资源分配。
Materials
| Chernin's balanced salt solution (CBSS+) | For 1L of solution | ||
| 2.8 g sodium chloride | Fisher Scientific | S271-3 | Dissolve salts, except calcium chloride, and |
| 0.15 g of potassium chloride | Sigma-Aldrich | P5405 | sugars in 800 mL ddH2O |
| 0.07 g sodium phosphate, dibasic anhydrous | Fisher Scientific | S374-500 | Dissolve calcium chloride separately in 200 |
| 0.45 g magnesium sulfate heptahydrate | Sigma-Aldrich | M1880 | mL ddH2O |
| 0.53 g calcium chloride dihydrate | Mallinckrodt | 4160 | Slowly add calcium soln to the salt/sugar soln with |
| 0.05 g sodium bicarbonate | Fisher Scientific | S233-3 | with constant mixing |
| 1 g glucose | MP Biomedicals | 152527 | Adjust to pH 7.2 and filter sterilize using a |
| 1 g trehalose | Sigma-Aldrich | T0167 | 0.22 µm disposable bottle-top filter |
| 10 mL 100X penicillin/streptomycin | Hyclone | SV30010 | Add filtered penicillin and streptomycin soln prior use |
| Incomplete Bge medium (Ibge) | For 900 mL solution | ||
| 220 mL Schneider’s Drosophila medium modified | Lonza | 04-351Q | Mix Schneider's medium with 680 mL ddH2O |
| 4.5 g lactalbumin enzymatic hydrolysate | Sigma-Aldrich | L9010 | Add lactalbumin hydrolysate and galactose |
| 1.3 g galactose | Sigma-Aldrich | G0625 | Adjust to pH 7.2 and filter sterilize using a 0.22 µm pre-sterilized disposable bottle-top filter |
| Complete Bge medium (cBge) | For 100 mL of solution | ||
| 90 mL Incomplete Bge medium | To heat-inactivate FBS: Incubate thawed FBS in | ||
| 9 mL heat-inact. fetal bovine serum (FBS) (Optima) | Atlanta Biologicals | S12450 | waterbath at 60°C for 1 hr while gently |
| 1 mL 100X penicillin/streptomycin | Hyclone | SV30010 | swirling the bottle every 10 min Aliquot heat-inactivated FBS into 15-mL tubes and store at -20°C. Mix medium + FBS and filter sterilize using a 0.22 µm pre-sterilized disposable bottle-top filter Add penicillin and streptomycin prior to use |
| Pond water (stock solution) | 1L of stock solution | ||
| 12.5 g calcium carbonate | Fisher Scientific | C64-500 | Mix all salts in 1L of ddH2O |
| 1.25 g magnesium carbonate | Fisher Scientific | M27-500 | Note that the salts will not have completely |
| 1.25 g sodium chloride | Fisher Scientific | S271-3 | dissolved. Shake vigorously to suspend |
| 0.25 g potassium chloride | Sigma-Aldrich | P5405 | salts prior to making the working soln |
| Pond water (working solution) | 1.5L of solution | ||
| 0.8 mL stock solution pond water (shake prior use) in | Mix stock to ddH2O | ||
| 1500 mL of ddH2O | Sterilize pond water by autoclaving (slow cycle) | ||
| 1.5 mL of 100X penicillin/streptomycin | Hyclone | SV30010 | Add penicillin and streptomycin prior use |
| Saline solution (1.2% NaCl) | 1.5L of solution | ||
| 18 g sodium chloride in 1500 mL of ddH2O | Fisher Scientific | S271-3 | Autoclave saline solution to sterilize |
| 1.5 mL of 100X penicillin/ streptomycin | Hyclone | SV30010 | Add penicillin and streptomycin prior use |
| Additional equipment and material: | |||
| 7-L mouse euthanizing chamber | Following approved IACUC protocol no. V001551 | ||
| Mice | Taconic Biosciences | Swiss-Webster, female, 6-wk old, murine | |
| CO2 tank and regulator | pathogen-free | ||
| 24-well tissue culture plate | TPP | 92424 | |
| 1-L volumetric flasks | |||
| Light source (150W) | Chiu Tech Corp | Model F0-150 | |
| Centrifuge, refrigerated, swinging bucket | Eppendorf | Model 5810R | |
| Centrifuge bottles (250 mL) | Nalgene | ||
| 15-mL centrifuge tubes, sterile | Corning | 430053 | |
| Sterile disposable transfer pipets | Fisher Scientific | 1371120 | |
| 0.22 µm pre-sterilized disposable bottle-top filter | EMD Millipore | SCGPS05RE | |
| Stainless steel blender | Waring Commercial | Model 51BL31 | |
| Blender cup, 100 mL capacity | Waring Commercial | ||
| Inverted compound microscope | Nikon Instruments | Eclipse TE300 |
References
- Colley, D. G., Bustinduy, A. L., Secor, W. E., King, C. H. Human schistosomiasis. Lancet. 383, 2253-2264 (2014).
- WHO. Schistosomiasis: number of people treated worldwide in 2013. Wkly. Epidemiol. Rec. 5 (90), 25-32 (2015).
- Yoshino, T. P., Gourbal, B., Théron, A., Jamieson, B. G. M. Schistosoma sporocysts. Schistosoma: biology, pathology and control. , 118-148 (2017).
- Bayne, C. J. Successful parasitism of vector snail Biomphalaria glabrata by the human blood fluke (trematode) Schistosoma mansoni: a 2009 assessment. Mol. Biochem. Parasitol. 165 (1), 8-18 (2009).
- Yoshino, T. P., Coustau, C., Toledo, R., Fried, B. Immunobiology of Biomphalaria-trematode interactions. Biomphalaria snails and larval trematodes. , 159-189 (2011).
- Coustau, C., et al. Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade. Fish Shellfish Immunol. 46 (1), 5-16 (2015).
- Bayne, C. J., Hahn, U. K., Bender, R. C. Mechanisms of molluscan host resistance and of parasite strategies for survival. Parasitology. 123, S159-S167 (2001).
- McMullen, D. B., Beaver, P. C. Studies on schistosome dermatitis. IX. The life cycles of three dermatitis-producing schistosomes from birds and a discussion of the subfamily Bilharziellinae (Trematoda: Schistosomatidae). Amer. J. Hyg. 42, 128-154 (1945).
- Voge, M., Seidel, J. S. Transformation in vitro of miracidia of Schistosoma mansoni and S. japonicum into young sprocysts. J. Parasitol. 58 (4), 699-704 (1972).
- Eloi-Santos, S., Olsen, N. J., Correa-Oliveira, R., Colley, D. G. Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice. Exp. Parasitol. 75 (2), 168-175 (1992).
- Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Freitas, T. C., Liang, Y. S. Schistosomiasis. Curr Proto Immunol. 103, 19.1:19.1.1-19.1.58 (2013).
- Basch, P. F., DiConza, J. J. The miracidium-sporocyst transition in Schistosoma mansoni: surface changes in vitro with ultrastructural correlation. J. Parasitol. 60 (6), 935-941 (1974).
- Hansen, E. L. Secondary daughter sporocysts of Schistosoma mansoni: their occurrence and cultivation. Ann. N. Y. Acad. Sci. 266, 426-436 (1975).
- Stibbs, H. H., Owczarzak, O., Bayne, C. J., DeWan, P. Schistosome sporocyst-killing amoebae Isolated from Biomphalaria glabrata. J. Invertebr. Pathol. 33, 159-170 (1979).
- DiConza, J. J., Basch, P. F. Axenic cultivation of Schistosoma mansoni daughter sporocysts. J. Parasitol. 60 (5), 757-763 (1974).
- Hansen, E. L., Maramorosch, K. A cell line from embryos of Biomphalaria glabrata (Pulmonata): Establishment and characteristics. Invertebrate tissue culture: Research applications. , 75-97 (1976).
- Yoshino, T. P., Laursen, J. R. Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells. J. Parasitol. 81 (5), 714-722 (1995).
- Ivanchenko, M. G., et al. Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni. Proc. Natl. Acad. Sci. USA. 96, 4965-4970 (1999).
- Yoshino, T. P., Bayne, C. J., Bickham, U. Molluscan cells in culture: primary cell cultures and cell lines. Can. J. Zool. 91, 391-404 (2013).
- Coustau, C., Yoshino, T. P. Flukes without snails: Advances in the in vitro cultivation of intramolluscan stages of trematodes. Exp. Parasitol. 94, 62-66 (2000).
- Milligan, J. N., Jolly, E. R. Cercarial transformation and in vitro cultivation of Schistosoma mansoni schistosomules. J. Vis. Exp. (54), e3191 (2011).
- Basch, P. F. Cultivation of Schistosoma mansoniin vitro. II production of infertile eggs by worm pairs cultured from cercariae. J. Parasitol. 67 (2), 186-190 (1981).
